Reverse transcription-quantitative PCR (RT-qPCR) for detection of miR-221 in lung tissue

Total RNA was extracted from lung tissue using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.). Briefly, a 100 mg sample was added to 1 ml TRIzol and homogenized. Subsequently, cDNA was synthesized according to the PrimeScript RT reagent kit instructions (Takara Bio, Inc.). cDNA (2 µl) was used as a template and amplification was carried out according to the RT-qPCR kit instructions (Invitrogen; Thermo Fisher Scientific, Inc.). Primer sequences are shown in Table II. The relative changes in mRNA expression levels were calculated using the 2−ΔΔCq Method (38). The reaction conditions were as follows: Pre-denaturation at 95°C for 5 min, followed by 30 cycles at 94°C for 30 sec, 55°C for 30 sec and 72°C for 60 sec, and a final extension step at 72°C for 1 min.

miR-221 and U6 primer sequences.

miR-221, microRNA-221.

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